Genome Sequence of Shigella flexneri Serotype 5a Strain M90T Sm

Abstract

Bacteria of the genusT he genus Shigella comprises 4 species, including Shigella flexneri, which is comprised of 14 different serotypes and subserotypes (2). S. flexneri strain M90T Sm is commonly used in the laboratory to understand the molecular basis of shigellosis (1,5,9).Two amplification-free libraries were constructed from 5 g of M90T Sm genomic DNA each, and the genome was sequenced using a 2-by-110-base paired-end strategy on the Illumina HiSeq2000 platform. Libraries were constructed using the NEBNext DNA sample prep master mix set 1 (NEB; part number E6040L) and barcoded in an overnight ligation. Libraries were size selected from 450 bp to 500 bp, quantified by quantitative PCR (qPCR) (Kapa BioSystems), pooled, requantified, and clustered using cBot on a single lane with TruSeq PE cluster kit v2 chemistry. A total of 129,937,458 single-end reads were filtered by demultiplexing using two indexes (AGTCGATC/CAGTCTGA) and the signal purity filter. The resulting 73,325,436 reads were trimmed to 54 bp, the point at which the mean quality score dropped below 30 (Illumina 1.5ϩ phred quality score), using the Fastx toolkit (version 0.0.13) (http://hannonlab.cshl.edu/fastx_toolkit/index .html). Half of the preprocessed reads (34,231,825 reads) were mapped onto the chromosomal sequence of S. flexneri serotype 5b strain 8401 (GenBank accession no. NC_008258) (6) using the Burrows-Wheeler aligner (BWA) (version 0.5.9) (3). A total of 28,876,862 reads (84.3%) were successfully mapped to a depth of 340ϫ. Single nucleotide polymorphism (SNP) calls and consensus sequence generation were performed using the pileup function in SAMtools (version 0.1.16) (4). The integrated genomics viewer (IGV) (version 1.5) (7) was used to examine regions of low coverage (0ϫ to 160ϫ) and/or low quality. A total of 39 positions within the single-copy region of the genome were verified by Sanger sequencing. The remaining 119 gaps were within multicopy regions, such as insertion sequences (IS), ribosomal DNAs, and the tufA gene. The GϩC content was calculated using Artemis (version 13.0) (8).The sequence of the S. flexneri M90T Sm genome covers 99.9% of the 4,574,284-bp chromosome of S. flexneri 8401 (6). A total of 1,433 SNPs and 176 indels were identified. About one-third of the SNPs (543 sites) cause nonsynonymous substitutions in S. flexneri M90T Sm. Twenty-one indels in M90T Sm cause frameshifts, and 9 indels correct 9 frame-shifted genes in S. flexneri 8401. A tandem repeat of tRNAs (tRNA-Lys and tRNA-Val) and one hypothetical protein that both do not exist in S. flexneri 8401 were found in M90T Sm. The M90T Sm genome contains 3,676 open reading frames (ORFs) (excluding IS ORFs), 99 tRNA genes, and 22 rRNA genes. The sequence has an average GϩC content of 50.9% in its entirety with a GϩC content of 52% within coding genes.The S. flexneri M90T Sm genome will enhance ongoing wholegenome comparisons across all S. flexneri serotypes for a fuller understanding of the evolution of S. flexneri and provides accurate sequence information need...

Genomic Portrait of the Evolution and Epidemic Spread of a Recently Emerged Multidrug-Resistant Shigella flexneri Clone in China

“…In order to elucidate the genomic basis of the epidemic spread of S. flexneri serotype Xv and the endemicity of shigellosis in China, we sequenced 59 S. flexneri isolates, of which 50 were ST91 and 25 were serotype Xv, from different regions of China, covering 10 years from 1997 to 2006 ( GenBank accession no. CM001474.1]) were included for comparison (9,(33)(34)(35)(36). A final set of 1,790 chromosomal SNPs in the core genome regions, excluding SNPs in repetitive or mobile sequences, were identified from a total of 64 genomes.…”

“…Among the 34 SNPs, 12, 13, and 9 were noncoding SNPs (ncSNPs), synonymous SNPs (sSNPs), and nonsynonymous SNPs (nsSNPs), respectively. Lineage II contained strains 2457T (serotype 2a, ST86) and M90T (serotype 5a, ST144), which were isolated in Japan (in 1954) and France (year of isolation unknown), respectively; both strains have been extensively used in laboratory experiments (34,(36)(37)(38) …”

Virulent Shigella flexneri Affects Secretion, Expression, and Glycosylation of Gel-Forming Mucins in Mucus-Producing Cells

“…As such, because sulfated glycans are ligands for only a few bacterial species (74), the appearance of sulfated glycans upon an infectious process can be seen as an advantage for the colonization of pathogenic bacteria expressing glycosulfatases by favoring establishment of their own niche to the detriment of species that are not equipped with such degradative enzymes. Interestingly, this could be the strategy used by S. flexneri, whose genome sequence reveals the existence of at least three genes encoding putative sulfatases, named yejM, yidJ, and ORF1680 (75).…”

Shigella Outer Membrane Protein PSSP-1 Is Broadly Protective against Shigella Infection

“…S. flexneri serotype 2a strain 2457T (12), serotype 5a strain M90T (13), and serotype 6 (14), S. boydii (4), S. dysenteriae serotype 1 strain 10398 (15), and S. flexneri serotype 2a vaccine strain SC602 (16) were used in this study. For intranasal (i.n.)…”